4.1. Sample characteristics
Sixty batches of WLZs were collected from the Shanxi-1 (Shānxi), Shanxi-2 (Shănxi), Hubei, and Hebei provinces of China, which are the major production areas for WLZs. Samples W1-W11were collected from Shanxi-1 province; W12-W21 were collected from Shanxi-2 province; W22-W28 were collected from the same manufacturer in QL city, Shanxi-2 province; W29-W33 collected from Hubei province; W34-W35, in which W34 was collected from Hubei province in 2010 and W35 was collected from Shanxi-2 province in 2012. V1, V4-V8, V13, V18-V19, and V22-V23 were collected from Shanxi-2 province; V2, V9-V12, V20-V21, and V24-V25 were collected from Shanxi-1 province; and V3 and V14-V17 were collected from Hebei province.
4.2. Intuitionistic analysis of elemental composition of WLZs
A summary of the concentrations of the 30 elements in WLZs and Cacumen Platycladi is shown in the Appendix. A. Considerable attention has been paid to pre-analytical methods. Among them, when more than 60% of the samples showed concentrations below LOQs, the result was given as an estimate rather than a value (the reference values proposed were only indicative, as below the LOQ). Twenty-nine elements (excluding Be) showed 100% of measured values above the LOQs, whereas Be showed 100% below the LOQ. In our method, 77Se and 114Cd isotopes were chosen because they are more abundant than 82Se and 111Cd isotopes. The mean values, median line, outlier data, and concentration range (25–75% and 1.5 folds of Inter-Quartile Range) of all investigated elements are shown in Fig. 2. Hg, a toxic element, was analyzed separately because of its extremely low content. The concentration ranges of the studied elements were observed. Notably, the order of elemental content in Cacumen Platycladi was consistent with that of WLZ. The detailed analysis is as follows.
4.2.1. Major elements
The major essential elements investigated were Na, Mg, K, Ca, and P; the sum of these seven major elements accounted for nearly 99% of the total investigated elements. Unlike other trace elements, Al and Fe are at a “macro” level in both WLZ and V-WLZ, therefore we classified Fe and Al as major elements for further analysis. According to the results depicted in Fig. 2, the major elements were found in the decreasing order of K > Mg > Ca > P ≈ Al > Fe > Na in WLZ, whereas the order of element mean concentrations in V-WLZ was K > Mg > Ca > P ≈ Al ≈ Na > Fe.
K was the most abundant macro-element of WLZs, accounting for approximately 46.66% and 44.41% of all analyzed elements in WLZ and V-WLZ, respectively. Mg was the second most abundant macro-element, representing 14.51% and 14.07% of the total investigated elements, respectively. Hence, we can speculate that the reason why the K content was the highest could be related to K being a product of the digestive tract. The mean concentration of Na was significantly increased after being processed with vinegar, varying from 2.26% (WLZ) to 7.71% (V-WLZ). Additionally, the average concentration of Ca and Al were reduced, ranging from 11.86% and 9.71% (WLZ) to 10.74% and 8.33% (V-WLZ), respectively. The concentration of Fe displayed similar levels for WLZ and V-WLZ with a value of 5.44% and 5.48%, respectively. Generally, Fe levels in traditional Chinese medicines used for invigorating blood are high.
4.2.2. Trace elements
The current Chinese pharmacopeia method for monitoring inorganic contaminants in pharmaceutical samples is defined in the general chapter < 2321>, including the elements Hg, Pb, As, Cd, and Cu. In contrast to other trace elements, these five heavy metals are potentially toxic for human health and their overdose may cause diseases. Consequently, we classified the trace elements as essential and potentially toxic for further discussion.
Essential elements
Compared to major elements, essential trace metals reliably reflect the feed strategy and environment of Trogopterus xanthipes, although their total content is less than 1% of all the analyzed elements. The essential trace elements were divided into three gradients according to their concentrations. In the first gradient (40–140 mg/kg), the average concentrations decreased in the order Mn > Sr > Zn ≈ Ba in both WLZs. Fe, Zn, Mn, and Cu have critical functional roles in hematopoiesis, immunity, and bone metabolism and might have direct and indirect effects on bone cells or bone mineralization.
The second gradient included the elements Rb, Cr, Ni, V, Se, Co, Ga, Cs, and Sb, with mean concentrations ranging from 0.2–10 mg/kg. The third gradient, including Ag, Be, Tl, Sn, and U, resulted in the less abundant trace essential elements with comparatively lower levels, and the mean concentrations were less than 0.2 mg/kg, which suggests a negligible role of WLZs as a medicinal source of Ag, Be, Tl, Sn, and U.
Toxic elements
The presence of potentially toxic metals in WLZs typically reflects an exogenous influence that may be correlated with dietary sources and environmental pollution at the feeding sites. According to the Chinese pharmacopeia 2020, Hg, Pb, As, Cd, and Cu are toxic heavy metals that can be detrimental to human health if they exceed certain limits. However, there are no limit standards for toxic elements in animal fecal drugs in the pharmacopeia. The permissible limits of some elements in herbal medicines are given in the Chinese pharmacopeia 2020 as reference values. The concentrations of Pb, Cd, As, Hg, and Cu were 5, 1, 2, 0.2, and 20 mg/kg, respectively.
These elements were found in decreasing order of Pb ≈ Cu > As > Cd > Hg in both WLZs. The content of Hg in WLZ and V-WLZ was below 0.06 mg/kg (0.010–0.058 mg/kg), which indicates the low intake of Hg from diet and atmosphere. According to the reference values proposed by the Chinese pharmacopeia (2020), the sample Cd concentrations were within the prescribed limits in our study, ranging from 0.098 to 0.534 mg/kg. Almost all sample concentrations for As were within the normal value; only five samples were more than 2 mg/kg (2.049–5.840 mg/kg). The Pb content in almost all samples was present at elevated concentrations, ranging from 3 to 20 mg/kg. The results showed that the Pb contents in some samples were outside the limits. Moreover, we found a significant difference in Pb content (0.798–15.366 mg/kg) in Cacumen Platycladi; therefore, we speculate that the relatively high concentration found in the samples could be related to the consumption of Cacumen Platycladi. Consequently, toxic elements accumulated in plants can also threaten human health through the food chain. Furthermore, the toxicity of metallic and non-metallic elements in a drug depends on the chemical form and ligand complex in which they are present.
4.3. Correlation analysis
Pearson’s correlation analysis results of the total element contents in WLZ, V-WLZ, and Cacumen Platycladi are shown in Fig. 3. Inter-element relationships provide important information on their sources, which can indicate whether there is a synergistic or antagonistic relationship between different elements. If r > 0 (< 0), there is a probable positive (negative) correlation; if r = 0, there is no relationship. Moreover, if r > 0.5 (< -0.5), the positive (negative) correlation is said to be strong, and weak when r < 0.5 (> -0.5).
As depicted in Fig. 3, there were robust positive correlations (r > 0.7, p < 0.01) among the ten elements, including U, Tl, Cr, Ni, Cs, Fe, Al, Co, Se, Ga, V, and As, in both WLZ and V-WLZ. A strong association (r > 0.35, p < 0.05) between Cu, Rb, Mg, Pb, Sb, Ag, and the above ten elements (except for As) are also shown in Fig. 3 (a) and (b). Although there is a slight difference in the correlation between the above four elements in WLZ and V-WLZ, the metal pairs between Cu, Rb, Mg, and Pb have probable positive correlations (r > 0.2) except for Cu-Mg (0.07) in V-WLZ. Additionally, significant positive correlations at the 95% confidence level were observed between K-P-Mn-Cu-Sr-Rb-Mn, Mg-Ca, Hg-Zn-Cd-Pb-Ba-Ag in both WLZ and V-WLZ, whereas significant positive correlations between other elements were only present in V-WLZ, such as Ba-(Cd, Zn, U, Co, Tl, Ag, Se, Ga), P-(Hg, Zn, K), Mn-(Sb, Cd, Zn), Sr-(U, Ag, Hg, Cd, Zn, Sn), K-(Cd, Al), and Sn-(Sb, Pb, Zn). In addition, significant positive correlations, including Sr-(Tl, K), Mn-(P, Tl), and Ca-Al, only existed in WLZ. Negative and significant correlations between the metal pairs were observed only in WLZ, whereas K-Na, P- (Ca, Al, Mg) were observed in V-WLZ. Furthermore, there were positive correlations between Cs, Cr, Se, V, Fe, U, Al, Tl, As, Sb, Pb, Hg, and Cd in Cacumen Platycladi, which have substantial similarities with WLZs. However, further negative correlations were observed in Cacumen Platycladi, including K- (Cs, Cr, Fe, V, Sb) and Ca-As.
Hence, we can conclude that, the high correlation coefficients between the above elements (U, Tl, Cr, Ni, Cs, Fe, Al, Co, Se, Ga, V, As, Cu, Rb, Mg, and Pb) indicate that the increase (decrease) of one of the elements is associated with the increase (decrease) of other elements, which helps in controlling toxic elements to a certain extent. Moreover, compared with WLZ, the positive/negative correlations between partial elements in V-WLZ are more or less enhanced. The significant similarities and differences between WLZs and Cacumen Platycladi could be related to the digestion and metabolism of Trogopterus xanthipes.
Figure 3 Correlation analysis of element content in WLZ, V-WLZ, and Cacumen Platycladi (Pearson, *p < 0.05, **p < 0.01)
4.4. PCA analysis
The PCA bilinear model constructed for variables gave an interpretable overview of the information on the most meaningful parameters that describe the whole multi-dimensional data set and enabled data reduction with a minimum loss of original [27, 28]. It allowed a reduction of twenty-nine variables (the element below LOQ was excluded) to six/five principal components (PC1-PC6)/(PC1-PC5) in WLZ and V-WLZ respectively, which were distinguished as important factors according to the eigenvalue-one-criterion (their eigenvalues were above 1). From Tables 5 and 6, we can see that these six/five PCs explained approximately 85% of the total variance, which was sufficient to describe the whole WLZs.
Figure 4a shows that the PC1 explained more than 43.10% of the total variance and revealed an apparent clustering between elements (U, Tl, Cs, Ga, Co, As, Se, V, Ni, Cr, Fe, and Al), which were characterized by the high positive loading values on PC1. The PC2 was associated with positively correlated Zn, Hg, P, K Ag, Ba, Pb, Cu, and Cd and provided information enclosed in the next 12.69% of the total variance. However, major elements (Na, Mg, and Ca) had low positive loading values on PC1-2, suggesting individualized behavior. Figure 5a shows that most elements were positively correlated with PC1 and only Hg, P, and K were negatively correlated with PC. A strong association between Tl, U, Cs, Se, Co, Ga, V, Ni, Al, Cr, Cu, Fe, and Pb is also observed because they presented high positive loading values for PC1. The contribution of the second factor was 18.9%, which showed high loading values for Zn, Cd, Hg, K, Sr, Pb, Ba, and Mn, with weak loading values for Ag, P, and Sn. These results indicate that the correlations between the elements changed after processing with vinegar.
Figure 4b and 5b show a graphic distribution of WLZ and V-WLZ (sampling sites) analyzed according to their component scores (PC1 vs. PC2). The samples on the right-hand side, with positive PC1 values were characterized by higher concentrations of Tl, U, Cs, Se, Co, Ga, V, Ni, Al, Cr, Cu, Fe, and Pb. The samples in the top part of the plot, with positive PC2 values featured higher concentrations of Zn, Cd, Hg, K, Pb, Ba, and Mn. Therefore, by overlapping the loading and score plots, it is evident that there is an apparent clustering among Group C in WLZ, with negative values of PC1 and PC2, which featured the lowest concentration levels of almost all elements. This could be because the samples in Group C were bathed and unprocessed.
Moreover, it can be interpreted that some samples in Groups A, B, and D have strong commonalities and show lower concentration levels because their sites plot on the negative side of the PC score plot. However, samples further showed different individualities from the same site, mainly due to the different ways in which humans raise Trogopterus xanthipes. Notably, Group E, including W34 and W35, was significantly different from the other samples. Remarkably, the heavy metals Hg, Ag, Cd, Pb, Ba, and Zn showed the highest concentrations in W35, which can be interpreted as being closely related to the ages of WLZ. Hence, it is necessary to specify the shelf life of traditional Chinese medicine.
Table 5
Eigenvalue and contribution in WLZ
| Principal Component Number | Eigenvalue | Percentage of Variance | Cumulative (%) |
| PC1 | 12.49 | 43.101 | 43.101 |
| PC2 | 3.68 | 12.69 | 55.79 |
| PC3 | 2.83 | 9.75 | 65.54 |
| PC4 | 2.77 | 9.57 | 75.10 |
| PC5 | 1.82 | 6.29 | 81.40 |
| PC6 | 1.26 | 4.34 | 85.73 |
Table 6
Eigenvalue and contribution in V-WLZ
| Principal Component Number | Eigenvalue | Percentage of Variance | Cumulative (%) |
| PC1 | 12.54 | 43.23 | 43.23 |
| PC2 | 5.48 | 18.91 | 62.14 |
| PC3 | 2.80 | 9.65 | 71.79 |
| PC4 | 2.09 | 7.20 | 78.99 |
| PC5 | 1.65 | 5.68 | 84.68 |
4.5. MPI and H-MPI
The MPI was calculated to examine the overall metal content in the studied WLZs, excluding the non-metal elements P, As, and Se. The H-MPI was calculated to examine the heavy metal (except for P, As, Se, K, Ca, Na, Mg, and Al) contents in the studied WLZs. This method can comprehensively reflect the levels of bioaccumulation of heavy metals in WLZ and V-WLZ and can be further used to evaluate the degree of heavy metal pollution of the organism.
A significant fluctuation range (4.129 to 20.980) was observed in WLZ (Fig. 6). Meanwhile, H-MPI varied over a large range (0.997 to 6.291) in WLZ, demonstrating that there are relatively major differences in elemental contents between WLZ from different sampling spots. Moreover, the specimens W34 and W35 exhibited the highest values of H-MPI and MPI, which is related to the ages of WLZ and has an extent of elemental accumulation; however, the mechanism of this hypothesis needs further research to be proved.
On the contrary, the MPI varied within a comparatively narrow range (5.963 to 13.307) in V-WLZ. Therefore, we can speculate that the levels of metals in V-WLZ changed after processing with vinegar. Compared with WLZ, the average value of MPI (8.643) in V-WLZ is higher than that of WLZ (8.403); the reason could be that the vinegar processing adds other metal elements, such as Na, K, Fe, and Ca. The average value of H-MPI (2.030) in V-WLZ showed a slight decrease compared to that in WLZ (2.096); the lower H-MPI indicated that the samples were more secure for human beings, given the heavy metal contents only. Thus, the decrease of some heavy metal elements after processing could serve as evidence for the “toxicity-reducing” mechanism.
4.6. Spectra of inorganic elements
4.6.1. Detection of outlier data
Some samples contained elements with extremely high abundances, as shown in the boxplot in Fig. 2. Taking Ba as an example, the maximum concentration was 494.31 mg/kg in WLZ, which was almost eight times the average concentration (57.25 mg/kg). This sample is an outlier. To obtain a more objective assessment of the average concentrations and content ranges for different elements and to balance the representativeness of the samples, we deleted two outlier samples from the sample data. The average concentrations of the 66 remaining samples, including 33 WLZ, 25 V-WLZ, and 8 Cacumen Platycladi, were calculated and are shown in Fig. 7.
4.6.2. Spectra of inorganic elements
Further statistical analyses were conducted to better understand the elemental characteristics of WLZs samples. Consequently, to establish the fingerprint of the inorganic elements, we synchronously reduced some elements (K, Ca, Mg, P, Al, Fe, Na, Mn, Sr, Zn, and Ba) at tremendously high concentrations of 1000x and obtained the same order of magnitude. The spectra of various sources of WLZs and Cacumen Platycladi are plotted in Fig. 7a-c. Additionally, the average contents of the studied elements in WLZ, V-WLZ, and Cacumen Platycladi were calculated and are shown in Fig. 7d.
The results in Fig. 7a-b show that the elemental spectra of WLZ and V-WLZ from different sources have similar peak shapes but different relative contents. The concentrations of K, Ca, Mg, P, Cr, V, Ni, Al, As, Fe, Cu, Se, Rb, and Pb varied dramatically, with relatively high contents of K, Ca, Mg, P, Al, Fe, and Cu. Moreover, Na contained in different V-WLZs changed significantly, except for the above elements; this could be closely related to processing with vinegar. This is because vinegar contains a large amount of Na, and the processing of different manufacturers is distinct. Additionally, as shown in Fig. 7, it is easy to find out that the inorganic element spectra of WLZs in the two varieties is consistent, and the concentration levels of most elements are similar. In contrast, the contents of K, Ca, (Na), Mg, P, Cr, V, Ni, Al, As, Fe, Cu, Se, Rb, and Pb showed significant variations in WLZ (V-WLZ). Thus, we can conclude that the composition distribution of inorganic elements in WLZs has a certain regularity, and that the inorganic element chromatograms of different sources and varieties of WLZs have outstanding features and consistency, which can be used as a reference index to distinguish other medicinal materials.
Furthermore, the results indicated that the concentrations of the studied elements in WLZs and Cacumen Platycladi had very similar trends, as depicted in Fig. 7d. We speculate that a close relationship may exist between WLZs and Cacumen Platycladi. Significantly higher sample levels of K, Mg, Na, P, Cr, V, Ca, Ni, Al, As, Fe, Cu, Rb, and Pb in WLZs could be explained to a certain extent by the continuous accumulation of these elements due to daily Cacumen Platycladi consumption. Several studies have shown that K, Ca, Mg, Cu, Zn, Fe, and Mn enhance the pharmacological effects of traditional Chinese medicine. In conclusion, by overlapping the PCA data and the inorganic element spectra, we identified Fe, Al, Cu, Se, Pb, Rb, V, K, P, Na, Cr, As, and Ni as the characteristic elements in WLZ and V-WLZ.